Biosynthetic Gene Pyramiding Leads to Ascorbate Accumulation with Enhanced Oxidative Stress Tolerance in Tomato

Abstract

Ascorbic acid (AsA) has high antioxidant activities, and its biosynthesis has been well studied by engineering of a single structural gene (SG) in staple crops, such as tomato (Solanum lycopersicum). However, engineering the AsA metabolic pathway by multi-SG for biofortification remains unclear. In this study, pyramiding transgenic lines including GDP-Mannose 3′,5′-epimerase (GME) × GDP-d-mannose pyrophosphorylase (GMP), GDP-l-Gal phosphorylase (GGP) × l-Gal-1-P phosphatase (GPP) and GME × GMP × GGP × GPP, were obtained by hybridization of four key genes to get over-expression transgenic plants (GME, GMP, GGP, and GPP) in tomato. Pyramiding lines exhibited a significant increase in total ascorbate in leaves and red fruits except for GGP × GPP. Expression analysis indicated that increased accumulation of AsA in pyramiding transgenic lines is due to multigene regulation in AsA biosynthesis. Substrate feeding in leaf and fruit suggested that AsA biosynthesis was mainly contributed by the d-Man/l-Gal pathway in leaves, while alternative pathways may contribute to AsA accumulation in tomato fruit. Pyramiding lines showed an enhanced light response, stress tolerance, and AsA transport capacity. Also, fruit shape, fruit size, and soluble solids were slightly affected by pyramiding. This study provides the first comprehensive analysis of gene pyramiding for ascorbate biosynthesis in tomato. SGs pyramiding promotes AsA biosynthesis, which in turn enhances light response and oxidative stress tolerance. Also, the data revealed an alternative ascorbate biosynthesis pathway between leaves and fruit of tomato.

Manipulation of Ascorbate Biosynthetic, Recycling, and Regulatory Pathways for Improved Abiotic Stress Tolerance in Plants

“…Knock-down of the GME gene in tomato by RNAi reduced ascorbate to 44% of wild-type levels, supporting its important role in L-galactose ascorbate biosynthesis [32]. Increased expression of the GME gene has increased ascorbate concentrations 1.4-to 1.9-fold in Arabidopsis [24,33,34], 1.4-to 1.8-fold in tomato [26,35], and 1.9-fold in rice [28]. Increased expression of the GMP + GME + GGP + GPP genes in tomato only increased ascorbate concentrations 2.0-fold, the same fold-change for increased expression of the GGP gene alone, suggesting that GME is not a major rate-limiting step in the L-galactose pathway in tomato [26].…”

“…Missense mutations in the Arabidopsis GMP gene reduced ascorbate to 25% of wild-type levels, supporting its important role in L-galactose ascorbate biosynthesis [21][22][23]. Increased expression of the GMP gene has increased ascorbate concentrations in a range of species, including 1.3-fold in Arabidopsis [24], 2.5-fold in tobacco [25], 1.4-to 1.7-fold in tomato (Solanum lycopersicum L.) [26,27], and 1.5-fold in rice [28] (Table 1). Increased co-expression of the GMP + GME genes in tomato increased ascorbate concentrations 2.0-fold, a greater increase than the individual genes alone, suggesting that GMP may act synergistically with GME to increase ascorbate concentrations in tomato [26].…”

“…Increased co-expression of the GMP + GME genes in tomato increased ascorbate concentrations 2.0-fold, a greater increase than the individual genes alone, suggesting that GMP may act synergistically with GME to increase ascorbate concentrations in tomato [26]. Increased co-expression of the GMP + GME + GGP + GPP genes in tomato also increased ascorbate concentrations 2.0-fold; however, this was the same fold-change for increased expression of the GGP gene alone, suggesting that GMP is not a major rate-limiting step in the L-galactose pathway in tomato [26]. This conclusion is also supported by other studies increasing the expression of the GMP gene in Arabidopsis and tobacco and increasing the co-expression of the GMP + GME genes in tobacco that did not report significant changes in ascorbate concentrations [29,30] (Table 1).…”

“…Increased expression of the GMP gene has increased ascorbate concentrations in a range of species, including 1.3-fold in Arabidopsis [24], 2.5-fold in tobacco [25], 1.4-to 1.7-fold in tomato (Solanum lycopersicum L.) [26,27], and 1.5-fold in rice [28] (Table 1). Increased co-expression of the GMP + GME genes in tomato increased ascorbate concentrations 2.0-fold, a greater increase than the individual genes alone, suggesting that GMP may act synergistically with GME to increase ascorbate concentrations in tomato [26]. Increased co-expression of the GMP + GME + GGP + GPP genes in tomato also increased ascorbate concentrations 2.0-fold; however, this was the same fold-change for increased expression of the GGP gene alone, suggesting that GMP is not a major rate-limiting step in the L-galactose pathway in tomato [26].…”

“…Increased expression of the GME gene has increased ascorbate concentrations 1.4-to 1.9-fold in Arabidopsis [24,33,34], 1.4-to 1.8-fold in tomato [26,35], and 1.9-fold in rice [28]. Increased expression of the GMP + GME + GGP + GPP genes in tomato only increased ascorbate concentrations 2.0-fold, the same fold-change for increased expression of the GGP gene alone, suggesting that GME is not a major rate-limiting step in the L-galactose pathway in tomato [26]. Moreover, increased expression of the GME gene in tobacco did not significantly change ascorbate concentrations, suggesting the GME is not a rate-limiting step of the L-galactose pathway in tobacco [29].…”